Osteoporosis currently affects millions of Americans and its prevalence is increasing as the aging population continues to rise. Since the osteoclast (OC) is the exclusive mediator of bone resorption, it is a primary therapeutic target for treatment and cure of this disease. Optimal bone resorption requires organization of the OC cytoskeleton, which is mediated by the av (33 integrin. Thus, discovering the mechanisms by which the integrin mediates organization of the OC cytoskeleton may provide novel anti-osteoporosis therapeutic targets. The SLP-76 adaptor protein is a link between plasma membrane receptors and cytoskeletal effector proteins in many cell types and we find that OCs lacking SLP-76 exhibit cytoskeletal abnormalities. Thus, we hypothesize that SLP-76 mediates adhesion-dependent cytoskeletal remodeling in the OC and our specific aim is to define the mechanism of SLP-76 mediated cytoskeletal reorganization in the OC in response to av(33 adhesion. Hence, we will determine the functional significance of SLP-76 in the OC in vitro (Sub-aim 1), identify the signals downstream of SLP-76 that mediate cytoskeletal reorganization (Subaim 2), and characterize the bone phenotype of SLP-76-/- mice in vivo (Sub-aim 3). To this end, we will utilize SLP-76 deficient mice as a source of bone marrow for growing mature OCs and use methods established previously in our lab for studying OC function. We will observe OC morphology and cytoskeletal structures (TRAP and FITC-phalloidin actin ring staining) and quantify levels of OC bone resorption in vitro (CTx assay and WGA-lectin-HRP staining). Subsequently, we develop various constructs of SLP-76 using site directed mutagenesis and retrovirally reconstitute SLP-76 deficient cells, in order to study the structure function relationship of SLP-76 and its binding effector proteins, using Western blot analysis as a read out. Lastly, we will examine the SLP-76 deficient mouse in vivo and determine its bone phenotype (using histomorphometry, DEXA, and microCT) under basal conditions, estrogen-deprivation (post-ovariectomy), and after injections with proteins that stimulate OC function (RANKL). In summary, we will determine the in vitro and in vivo function of SLP-76 and define the SLP-76 signaling pathway in OCs. The osteoclast is the cell responsible for bone loss and is thus a target for osteoporosis research. Our aim is to study how the osteoclast resorbs bone in the hope of elucidating new therapeutic targets for the prevention and treatment of osteoporosis.